Didn’t feel a thing: Novel CE-MS method for analyzing metabolites in live cells

Ezine

Published: May 15, 2017

Author: Jon Evans

Channels: Electrophoresis

Embryo cells.

Photo: E.C. Raff and R.A. Raff.

Single cells

Over the past few years, scientists such as Peter Nemes at the George Washington University in Washington, D.C., US, have shown how combinations of capillary electrophoresis and mass spectrometry (CE-MS) are now sensitive enough to analyze the contents of single cells. To get at those contents, though, the cells still have to be torn apart, which prevents any investigation into how those contents change over time in a live cell.

Now, Nemes and his colleagues have come up with a CE-MS method that can analyze the metabolites in a single cell without killing it. And to demonstrate their method’s abilities, they’ve applied it to a type of cell whose sole purpose is to change over time: embryo cells.

Their method begins by using a sharpened capillary with a 20μm tip to penetrate the outer membrane of an individual embryo cell and suck up a small quantity of cell material, around 10–15nL, comprising just 10% of the cellular volume. This extraction step is monitored in real-time using some form of optical microscopy, such as bright-field microscopy, and shouldn’t cause any major damage to the cell.

Frog embryo

Next, the cell material is deposited into a microvial and combined with 5μm of an aqueous organic solvent mixture to extract the metabolites. A 10nL sample of this extract is then injected into a specially-developed CE-MS system, with the two components linked by electrospray ionization.

For a first test of this method, Nemes and his colleagues used it on a frog embryo comprising just eight cells, comparing it to the CE-MS analysis of a dead frog embryo cell. As hoped, the process of extracting a small amount of material from one of the frog embryo cells didn’t seem to cause any harm, with the cell continuing to divide normally as the embryo grew to 16 cells.

Diluting the extracted cell material with the organic solvent mixture means that only 0.02% of the cell contents are actually analyzed by CE-MS, but the method still generated a similar spectra to that produced by analyzing the dead cell. Even though much more cell material was available for analysis with the dead cell. This allowed Nemes and his colleagues to detect 230 distinct peaks in the spectra derived from the live cell extract, from which they were able to identify 70 known metabolites. The peaks produced by the new method were also clearer than those generated from the dead cell, with much less background noise. This is probably because the process of extracting metabolites from a live cell collects far less interfering material such as salts and buffering agents from the cell culture media.

Metabolite changes

The new method was also much faster: it took over five minutes to kill an embryo cell and extract all its contents, while it took less than five seconds to extract the small amount of material from a living cell. This meant Nemes and his colleagues could extract material multiple times from the same cell before it divided, potentially providing a unique view on how the metabolites in a cell change over time. Again, this multiple sampling didn’t appear to cause the cell any harm, with the holes caused by the sharpened capillary quickly closing up.

Interestingly, the range and abundance of metabolites detected in a live cell were slightly different to that detected in the dead cell. Part of this probably reflects normal variation between cells, but it also likely reflects the fact that the process of killing a cell has a major effect on its metabolic state. This is supported by the finding of a higher concentration of metabolites known to be produced by oxidative stress in the dead cells than in the live ones.

To provide a really good demonstration of the benefits of their new CE-MS method, Nemes and his colleagues then used it to monitor the metabolites in a single cell and its descendents as a frog embryo grew from eight cells to 16 cells to 32 cells. This had never been done before and revealed significant changes in the concentration of 50 identified metabolites, offering unprecedented molecular insight into embryo development.

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